Nidoviruses (arteriviruses, coronaviruses, and roniviruses) are a phylogenetically compact but diverse group of positive-strand RNA viruses that includes important human and animal pathogens. Nidovirus RNA synthesis is mediated by a cytoplasmic membrane-associated replication/transcription complex that includes up to 16 viral nonstructural proteins (nsps), which carry common enzymatic activities, like the viral RNA polymerase, but also unusual and poorly understood RNA-processing functions. Of these, a conserved endoribonuclease (NendoU) is a major genetic marker that is unique to nidoviruses. NendoU activity was previously verified in vitro for the coronavirus nsp15, but not for any of its distantly related orthologs from other nidovirus lineages, like the arterivirus nsp11. Here, we show that the bacterially expressed nsp11 proteins of two arteriviruses, equine arteritis virus and porcine respiratory and reproductive syndrome virus, possess pyrimidine-specific endoribonuclease activity. RNA cleavage was independent of divalent cations in vitro and was greatly reduced by replacement of residues previously implicated in catalysis. Comparative characterization of the NendoU activity in arteriviruses and severe acute respiratory syndrome coronavirus revealed common and distinct features of their substrate requirements and reaction mechanism. Our data provide the first biochemical evidence of endoribonuclease activity associated with arterivirus nsp11 and support the conclusion that this remarkable RNA-processing enzyme, whose substrate in the infected cell remains to be identified, distinguishes nidoviruses from all other RNA viruses.
The replication/transcription complex of the arterivirus equine arteritis virus (EAV) is associated with paired membranes and/or double-membrane vesicles (DMVs) that are thought to originate from the endoplasmic reticulum. Previously, coexpression of two putative transmembrane nonstructural proteins (nsp2 and nsp3) was found to suffice to induce these remarkable membrane structures, which are typical of arterivirus infection. Here, site-directed mutagenesis was used to investigate the role of nsp3 in more detail. Liberation of the hydrophobic N terminus of nsp3, which is normally achieved by cleavage of the nsp2/3 junction by the nsp2 protease, was nonessential for the formation of DMVs. However, the substitution of each of a cluster of four conserved cysteine residues, residing in a predicted luminal loop of nsp3, completely blocked DMV formation. Some of these mutant nsp3 proteins were also found to be highly cytotoxic, in particular, exerting a dramatic effect on the endoplasmic reticulum. The functionality of an engineered N glycosylation site in the cysteine-containing loop confirmed both its presence in the lumen and the transmembrane nature of nsp3. This mutant displayed an interesting intermediate phenotype in terms of DMV formation, with paired and curved membranes being formed, but DMV formation apparently being impaired. The effect of nsp3 mutations on replicase polyprotein processing was investigated, and several mutations were found to influence processing of the region downstream of nsp3 by the nsp4 main protease. When tested in an EAV reverse genetics system, none of the nsp3 mutations was tolerated, again underlining the crucial role of the protein in the arterivirus life cycle.
Animal coronaviruses, such as infectious bronchitis virus (IBV), and arteriviruses, such as porcine reproductive and respiratory syndrome virus (PRRSV), are able to manifest highly contagious infections in their specific native hosts, thereby arising in critical economic damage to animal industries. This review discusses recent progress in studies of virus-host interactions during animal and human coronavirus and arterivirus infections, with emphasis on IBV-host cell interactions. These interactions may be directly involved in viral replication or lead to the alteration of certain signaling pathways, such as cell stress response and innate immunity, to facilitate viral replication and pathogenesis.
virus-host interactions; coronavirus; arterivirus
The gene segment encoding avian infectious bronchitis virus nonstructural protein 9 has been cloned and expressed in Escherichia coli. The protein has been crystallized and the crystals diffracted X-rays to 2.44 Å resolution.
Avian infectious bronchitis virus (IBV), which causes respiratory disease in infected birds, belongs to coronavirus group 3. IBV encodes 15 nonstructural proteins (nsp2–nsp16) which play crucial roles in RNA transcription and genome replication. Nonstructural protein 9 (nsp9) has been identified as a protein that is essential to viral replication because of its single-stranded RNA-binding ability. The gene segment encoding IBV nsp9 has been cloned and expressed in Escherichia coli. The protein has been crystallized and the crystals diffracted X-rays to 2.44 Å resolution. They belonged to the cubic space group I432, with unit-cell parameters a = b = c = 123.4 Å, α = β = γ = 90°. The asymmetric unit appeared to contain one molecule, with a solvent content of 62% (V
M = 3.26 Å3 Da−1).
infectious bronchitis virus; nonstructural proteins; coronaviruses
Two adjacent papainlike cysteine protease (PCP) domains, PCP alpha and PCP beta, were identified in the N-terminal region of the open reading frame 1a replicase proteins of the arteriviruses porcine reproductive and respiratory syndrome virus and lactate dehydrogenase-elevating virus. The replicase of the related virus equine arteritis virus contains only one active PCP in the corresponding region. Sequence comparison revealed that the equine arteritis virus PCP alpha counterpart probably was inactivated by loss of its catalytic Cys residue. For both porcine reproductive and respiratory syndrome virus and lactate dehydrogenase-elevating virus, the generation of two processing products, nsp1 alpha and nsp1 beta, was demonstrated by in vitro translation. Site-directed mutagenesis and sequence comparison were used to identify the putative active-site residues of the PCP alpha and PCP beta protease domains and to show that they mediate the nsp1 alpha/1 beta and nsp1 beta/2 cleavages, respectively.
The severe acute respiratory syndrome coronavirus (SARS-CoV) encodes proteins required for RNA transcription and genome replication as large polyproteins that are proteolytically processed by virus-encoded proteinases to produce mature replicase proteins. In this report, we generated antibodies against SARS-CoV predicted replicase protein and used the antibodies to identify and characterize 12 of the 16 predicted mature replicase proteins (nsp1, nsp2, nsp3, nsp4, nsp5, nsp8, nsp9, nsp12, nsp13, nsp14, nsp15, and nsp16) in SARS-CoV-infected Vero cells. Immunoblot analysis of infected-cell lysates identified proteins of the predicted sizes. Immunofluorescence microscopy detected similar patterns of punctate perinuclear and distributed cytoplasmic foci with all replicase antibodies and as early as 6 h postinfection. Dual-labeling studies demonstrated colocalization of replicase protein nsp8 with nsp2 and nsp3 in cytoplasmic complexes and also with LC3, a protein marker for autophagic vacuoles. Antibodies directed against mouse hepatitis virus (MHV) virions and against the putative RNA-dependent RNA polymerase (Pol) detected SARS-CoV nucleocapsid and nsp12 (Pol), respectively, in SARS-CoV-infected Vero cells. These results confirm the predicted protein processing pattern for mature SARS-CoV replicase proteins, demonstrate localization of replicase proteins to cytoplasmic complexes containing markers for autophagosome membranes, and suggest conservation of protein epitopes in the replicase and nucleocapsid of SARS-CoV and the group II coronavirus, MHV. Further, the results demonstrate the ability of replicase antibodies to detect SARS-CoV-infected cells as early as 6 h postinfection and thus represent important tools for studies of SARS-CoV replication, inhibition, and diagnosis.
The coronavirus nonstructural proteins (nsp's) derived from the replicase polyproteins collectively constitute the viral replication complexes, which are anchored to double-membrane vesicles. Little is known about the biogenesis of these complexes, the membrane anchoring of which is probably mediated by nsp3, nsp4, and nsp6, as they contain several putative transmembrane domains. As a first step to getting more insight into the formation of the coronavirus replication complex, the membrane topology, processing, and subcellular localization of nsp4 of the mouse hepatitis virus (MHV) and severe acute respiratory syndrome-associated coronavirus (SARS-CoV) were elucidated in this study. Both nsp4 proteins became N glycosylated, while their amino and carboxy termini were localized to the cytoplasm. These observations imply nsp4 to assemble in the membrane as a tetraspanning transmembrane protein with a Nendo/Cendo topology. The amino terminus of SARS-CoV nsp4, but not that of MHV nsp4, was shown to be (partially) processed by signal peptidase. nsp4 localized to the endoplasmic reticulum (ER) when expressed alone but was recruited to the replication complexes in infected cells. nsp4 present in these complexes did not colocalize with markers of the ER or Golgi apparatus, while the susceptibility of its sugars to endoglycosidase H indicated that the protein had also not traveled trough the latter compartment. The important role of the early secretory pathway in formation of the replication complexes was also demonstrated by the inhibition of coronaviral replication when the ER export machinery was blocked by use of the kinase inhibitor H89 or by expression of a mutant, Sar1[H79G].
The involvement of host proteins in the replication and transcription of viral RNA is a poorly understood area for many RNA viruses. For coronaviruses, it was long speculated that replication of the giant RNA genome and transcription of multiple subgenomic mRNA species by a unique discontinuous transcription mechanism may require host cofactors. To search for such cellular proteins, yeast two-hybrid screening was carried out by using the nonstructural protein 14 (nsp14) from the coronavirus infectious bronchitis virus (IBV) as a bait protein, leading to the identification of DDX1, a cellular RNA helicase in the DExD/H helicase family, as a potential interacting partner. This interaction was subsequently confirmed by coimmunoprecipitation assays with cells coexpressing the two proteins and with IBV-infected cells. Furthermore, the endogenous DDX1 protein was found to be relocated from the nucleus to the cytoplasm in IBV-infected cells. In addition to its interaction with IBV nsp14, DDX1 could also interact with the nsp14 protein from severe acute respiratory syndrome coronavirus (SARS-CoV), suggesting that interaction with DDX1 may be a general feature of coronavirus nsp14. The interacting domains were mapped to the C-terminal region of DDX1 containing motifs V and VI and to the N-terminal portion of nsp14. Manipulation of DDX1 expression, either by small interfering RNA-induced knockdown or by overexpression of a mutant DDX1 protein, confirmed that this interaction may enhance IBV replication. This study reveals that DDX1 contributes to efficient coronavirus replication in cell culture.
Coronavirus (CoV) 3C-like proteinase (3CLpro), located in nonstructural protein 5 (nsp5), processes the replicase polyproteins 1a and 1ab (pp1a and pp1ab) at 11 specific sites to produce 12 mature nonstructural proteins (nsp5 to nsp16). Structural and biochemical studies suggest that a conserved Gln residue at the P1 position is absolutely required for efficient cleavage. Here, we investigate the effects of amino acid substitution at the P1 position of 3CLpro cleavage sites of infectious bronchitis virus (IBV) on the cleavage efficiency and viral replication by in vitro cleavage assays and reverse genetic approaches. Our results demonstrated that a P1-Asn substitution at the nsp4-5/Q2779, nsp5-6/Q3086, nsp7-8/Q3462, nsp8-9/Q3672, and nsp9-10/Q3783 sites, a P1-Glu substitution at the nsp8-9/Q3672 site, and a P1-His substitution at the nsp15-16/Q6327 site were tolerated and allowed recovery of infectious mutant viruses, albeit with variable degrees of growth defects. In contrast, a P1-Asn substitution at the nsp6-7/Q3379, nsp12-13/Q4868, nsp13-14/Q5468, and nsp14-15/Q5989 sites, as well as a P1-Pro substitution at the nsp15-16/Q6327 site, abolished 3CLpro-mediated cleavage at the corresponding position and blocked the recovery of infectious viruses. Analysis of the effects of these lethal mutations on RNA synthesis suggested that processing intermediates, such as the nsp6-7, nsp12-13, nsp13-14, nsp14-15, and nsp15-16 precursors, may function in negative-stranded genomic RNA replication, whereas mature proteins may be required for subgenomic RNA (sgRNA) transcription. More interestingly, a mutant 3CLpro with either a P166S or P166L mutation was selected when an IBV infectious cDNA clone carrying the Q6327N mutation at the nsp15-16 site was introduced into cells. Either of the two mutations was proved to enhance significantly the 3CLpro-mediated cleavage efficiency at the nsp15-16 site with a P1-Asn substitution and compensate for the detrimental effects on recovery of infectious virus.
Generation of PI3P in the normally PI3P-deficient ER membrane makes the organelle a platform for autophagosome formation.
Autophagy is a catabolic process that allows cells to digest their cytoplasmic constituents via autophagosome formation and lysosomal degradation. Recently, an autophagy-specific phosphatidylinositol 3-kinase (PI3-kinase) complex, consisting of hVps34, hVps15, Beclin-1, and Atg14L, has been identified in mammalian cells. Atg14L is specific to this autophagy complex and localizes to the endoplasmic reticulum (ER). Knockdown of Atg14L leads to the disappearance of the DFCP1-positive omegasome, which is a membranous structure closely associated with both the autophagosome and the ER. A point mutation in Atg14L resulting in defective ER localization was also defective in the induction of autophagy. The addition of the ER-targeting motif of DFCP1 to this mutant fully complemented the autophagic defect in Atg14L knockout embryonic stem cells. Thus, Atg14L recruits a subset of class III PI3-kinase to the ER, where otherwise phosphatidylinositol 3-phosphate (PI3P) is essentially absent. The Atg14L-dependent appearance of PI3P in the ER makes this organelle the platform for autophagosome formation.
Coronaviruses are positive-strand RNA viruses that replicate in the cytoplasm of infected cells by generating a membrane-associated replicase complex. The replicase complex assembles on double membrane vesicles (DMVs). Here, we studied the role of a putative replicase anchor, nonstructural protein 4 (nsp4), in the assembly of murine coronavirus DMVs. We used reverse genetics to generate infectious clone viruses (icv) with an alanine substitution at nsp4 glycosylation site N176 or N237, or an asparagine to threonine substitution (nsp4-N258T), which is proposed to confer a temperature sensitive phenotype. We found that nsp4-N237A is lethal and nsp4-N258T generated a virus (designated Alb ts6 icv) that is temperature sensitive for viral replication. Analysis of Alb ts6 icv-infected cells revealed that there was a dramatic reduction in DMVs and that both nsp4 and nsp3 partially localized to mitochondria when cells were incubated at the non-permissive temperature. These results reveal a critical role of nsp4 in directing coronavirus DMV assembly.
coronavirus; nonstructural proteins; double membrane vesicles; ts mutant
Virus-induced membrane structures support the assembly and function of positive-strand RNA virus replication complexes. The replicase proteins of arteriviruses are associated with double-membrane vesicles (DMVs), which were previously proposed to derive from the endoplasmic reticulum (ER). Using electron tomography, we performed an in-depth ultrastructural analysis of cells infected with the prototypic arterivirus equine arteritis virus (EAV). We established that the outer membranes of EAV-induced DMVs are interconnected with each other and with the ER, thus forming a reticulovesicular network (RVN) resembling that previously described for the distantly related severe acute respiratory syndrome (SARS) coronavirus. Despite significant morphological differences, a striking parallel between the two virus groups, and possibly all members of the order Nidovirales, is the accumulation in the DMV interior of double-stranded RNA, the presumed intermediate of viral RNA synthesis. In our electron tomograms, connections between the DMV interior and cytosol could not be unambiguously identified, suggesting that the double-stranded RNA is compartmentalized by the DMV membranes. As a novel approach to visualize and quantify the RNA content of viral replication structures, we explored electron spectroscopic imaging of DMVs, which revealed the presence of phosphorus in amounts equaling on average a few dozen copies of the EAV RNA genome. Finally, our electron tomograms revealed a network of nucleocapsid protein-containing protein tubules that appears to be intertwined with the RVN. This potential intermediate in nucleocapsid formation, which was not observed in coronavirus-infected cells, suggests that arterivirus RNA synthesis and assembly are coordinated in intracellular space.
The open reading frame (ORF) 1b-encoded part of the equine arteritis virus (EAV) replicase is expressed by ribosomal frameshifting during genome translation, which results in the production of an ORF1ab fusion protein (345 kDa). Four ORF1b-encoded processing products, nsp9 (p80), nsp10 (p50), nsp11 (p26), and nsp12 (p12), have previously been identified in EAV-infected cells (L. C. van Dinten, A. L. M. Wassenaar, A. E. Gorbalenya, W. J. M. Spaan, and E. J. Snijder, J. Virol. 70:6625–6633, 1996). In the present study, the generation of these four nonstructural proteins was shown to be mediated by the nsp4 serine protease, which is the main viral protease (E. J. Snijder, A. L. M. Wassenaar, L. C. van Dinten, W. J. M. Spaan, and A. E. Gorbalenya, J. Biol. Chem. 271:4864–4871, 1996). Mutagenesis of candidate cleavage sites revealed that Glu-2370/Ser, Gln-2837/Ser, and Glu-3056/Gly are the probable nsp9/10, nsp10/11, and nsp11/12 junctions, respectively. Mutations which abolished ORF1b protein processing were introduced into a recently developed infectious cDNA clone (L. C. van Dinten, J. A. den Boon, A. L. M. Wassenaar, W. J. M. Spaan, and E. J. Snijder, Proc. Natl. Acad. Sci. USA 94:991–997, 1997). An analysis of these mutants showed that the selective blockage of ORF1b processing affected different stages of EAV reproduction. In particular, the mutant with the nsp10/11 cleavage site mutation Gln-2837→Pro displayed an unusual phenotype, since it was still capable of RNA synthesis but was incapable of producing infectious virus.
Arterivirus replicase polyproteins are cleaved into at least 13 mature nonstructural proteins (nsps), and in particular the nsp5-to-nsp8 region is subject to a complex processing cascade. The function of the largest subunit from this region, nsp7, which is further cleaved into nsp7α and nsp7β, is unknown. Using nuclear magnetic resonance (NMR) spectroscopy, we determined the solution structure of nsp7α of equine arteritis virus, revealing an interesting unique fold for this protein but thereby providing little clue to its possible functions. Nevertheless, structure-based reverse genetics studies established the importance of nsp7/nsp7α for viral RNA synthesis, thus providing a basis for future studies.
The positive-stranded RNA genome of the coronaviruses is translated from ORF1 to yield polyproteins that are proteolytically processed into intermediate and mature nonstructural proteins (nsps). Murine hepatitis virus (MHV) and severe acute respiratory syndrome coronavirus (SARS-CoV) polyproteins incorporate 16 protein domains (nsps), with nsp1 and nsp2 being the most variable among the coronaviruses and having no experimentally confirmed or predicted functions in replication. To determine if nsp2 is essential for viral replication, MHV and SARS-CoV genome RNA was generated with deletions of the nsp2 coding sequence (MHVΔnsp2 and SARSΔnsp2, respectively). Infectious MHVΔnsp2 and SARSΔnsp2 viruses recovered from electroporated cells had 0.5 to 1 log10 reductions in peak titers in single-cycle growth assays, as well as a reduction in viral RNA synthesis that was not specific for any positive-stranded RNA species. The Δnsp2 mutant viruses lacked expression of both nsp2 and an nsp2-nsp3 precursor, but cleaved the engineered chimeric nsp1-nsp3 cleavage site as efficiently as the native nsp1-nsp2 cleavage site. Replication complexes in MHVΔnsp2-infected cells lacked nsp2 but were morphologically indistinguishable from those of wild-type MHV by immunofluorescence. nsp2 expressed in cells by stable retroviral transduction was specifically recruited to viral replication complexes upon infection with MHVΔnsp2. These results demonstrate that while nsp2 of MHV and SARS-CoV is dispensable for viral replication in cell culture, deletion of the nsp2 coding sequence attenuates viral growth and RNA synthesis. These findings also provide a system for the study of determinants of nsp targeting and function.
Mouse hepatitis virus (MHV) is a member of the family Coronaviridae. These positive strand RNA viruses encode a replicase polyprotein that is processed into 16 nonstructural proteins (nsps). The nsps assemble with membranes to generate double membrane vesicles, which are the sites of viral RNA synthesis. MHV nsp3 contains multiple domains including two papain-like protease domains, PLP1 and PLP2, and a predicted transmembrane (TM) domain. In this study, we determined the membrane topology of nsp3-TM and showed that TM-mediated tethering of PLP2 is important for processing at cleavage site 3. Biochemical analysis revealed that nsp3 is an integral membrane protein that is inserted into endoplasmic reticulum (ER) membranes co-translationally and glycosylated at asparagine-2357. Proteinase K digestion experiments indicate that the TM domain of nsp3 has 4 membrane-spanning helices. We show that nsp3-TM is sufficient to mediate ER membrane association of a cytosolic protein. This study is the first detailed analysis of the topology and function of the coronavirus nsp3 TM domain.
coronavirus; replicase polyprotein; papain-like protease; transmembrane domain
The severe acute respiratory syndrome coronavirus (SARS-CoV) possesses a large 29.7-kb positive-stranded RNA genome. The first open reading frame encodes replicase polyproteins 1a and 1ab, which are cleaved to generate 16 “nonstructural” proteins, nsp1 to nsp16, involved in viral replication and/or RNA processing. Among these, nsp10 plays a critical role in minus-strand RNA synthesis in a related coronavirus, murine hepatitis virus. Here, we report the crystal structure of SARS-CoV nsp10 at a resolution of 1.8 Å as determined by single-wavelength anomalous dispersion using phases derived from hexatantalum dodecabromide. nsp10 is a single domain protein consisting of a pair of antiparallel N-terminal helices stacked against an irregular β-sheet, a coil-rich C terminus, and two Zn fingers. nsp10 represents a novel fold and is the first structural representative of this family of Zn finger proteins found so far exclusively in coronaviruses. The first Zn finger coordinates a Zn2+ ion in a unique conformation. The second Zn finger, with four cysteines, is a distant member of the “gag-knuckle fold group” of Zn2+-binding domains and appears to maintain the structural integrity of the C-terminal tail. A distinct clustering of basic residues on the protein surface suggests a nucleic acid-binding function. Gel shift assays indicate that in isolation, nsp10 binds single- and double-stranded RNA and DNA with high-micromolar affinity and without obvious sequence specificity. It is possible that nsp10 functions within a larger RNA-binding protein complex. However, its exact role within the replicase complex is still not clear.
Gene 1 of the coronavirus associated with severe acute respiratory syndrome (SARS) encodes replicase polyproteins that are predicted to be processed into 16 nonstructural proteins (nsps 1 to 16) by two viral proteases, a papain-like protease (PLpro) and a 3C-like protease (3CLpro). Here, we identify SARS coronavirus amino-terminal replicase products nsp1, nsp2, and nsp3 and describe trans-cleavage assays that characterize the protease activity required to generate these products. We generated polyclonal antisera to glutathione S-transferase-replicase fusion proteins and used the antisera to detect replicase intermediates and products in pulse-chase experiments. We found that nsp1 (p20) is rapidly processed from the replicase polyprotein. In contrast, processing at the nsp2/3 site is less efficient, since a ≈300-kDa intermediate (NSP2-3) is detected, but ultimately nsp2 (p71) and nsp3 (p213) are generated. We found that SARS coronavirus replicase products can be detected by 4 h postinfection in the cytoplasm of infected cells and that nsps 1 to 3 colocalize with newly synthesized viral RNA in punctate, perinuclear sites consistent with their predicted role in viral RNA synthesis. To determine if PLpro is responsible for processing these products, we cloned and expressed the PLpro domain and the predicted substrates and established PLpro trans-cleavage assays. We found that the PLpro domain is sufficient for processing the predicted nsp1/2 and nsp2/3 sites. Interestingly, expression of an extended region of PLpro that includes the downstream hydrophobic domain was required for processing at the predicted nsp3/4 site. We found that the hydrophobic domain is inserted into membranes and that the lumenal domain is glycosylated at asparagine residues 2249 and 2252. Thus, the hydrophobic domain may anchor the replication complex to intracellular membranes. These studies revealed that PLpro can cleave in trans at the three predicted cleavage sites and that it requires membrane association to process the nsp3/4 cleavage site.
The N-terminal domain of nsp2 from avian infectious bronchitis virus has been purified and crystallized. The crystals diffracted to 2.5 Å resolution.
Avian infectious bronchitis virus (IBV) is a prototype of the group III coronaviruses and encodes 15 nonstructural proteins which make up the transcription/replication machinery. The nsp2 protein from IBV has a unique and novel sequence and has no experimentally confirmed function in replication, whereas it has been proposed to be crucial for early viral infection and may inhibit the early host immune response. The gene that encodes a double-mutant IBV nsp2 N-terminal domain (residues 9–393 of the polyprotein, with mutations Q132L and L270F) was cloned and expressed in Escherichia coli and the protein was subjected to crystallization trials. The crystals diffracted to 2.5 Å resolution and belonged to space group P62 or P64, with unit-cell parameters a = b = 114.2, c = 61.0 Å, α = β = 90, γ = 120°. Each asymmetric unit contained one molecule.
coronaviruses; avian infectious bronchitis virus; nonstructural proteins; nsp2
Equine arteritis virus (EAV), the prototype Arterivirus, is a positive-stranded RNA virus that expresses its replicase in the form of two large polyproteins of 1,727 and 3,175 amino acids. The functional replicase subunits (nonstructural proteins), which drive EAV genome replication and subgenomic mRNA transcription, are generated by extensive proteolytic processing. Subgenomic mRNA transcription involves an unusual discontinuous step and generates the mRNAs for structural protein expression. Previously, the phenotype of mutant EAV030F, which carries a single replicase point mutation (Ser-2429→Pro), had implicated the nsp10 replicase subunit (51 kDa) in viral RNA synthesis, and in particular in subgenomic mRNA transcription. nsp10 contains an N-terminal (putative) metal-binding domain (MBD), located just upstream of the Ser-2429→Pro mutation, and a helicase activity in its C-terminal part. We have now analyzed the N-terminal domain of nsp10 in considerable detail. A total of 38 mutants, most of them carrying specific single point mutations, were tested in the context of an EAV infectious cDNA clone. Variable effects on viral genome replication and subgenomic mRNA transcription were observed. In general, our results indicated that the MBD region, and in particular a set of 13 conserved Cys and His residues that are assumed to be involved in zinc binding, is essential for viral RNA synthesis. On the basis of these data and comparative sequence analyses, we postulate that the MBD may employ a rather unusual mode of zinc binding that could result in the association of up to four zinc cations with this domain. The region containing residue Ser-2429 may play the role of “hinge spacer,” which connects the MBD to the rest of nsp10. Several mutations in this region specifically affected subgenomic mRNA synthesis. Furthermore, one of the MBD mutants was replication and transcription competent but did not produce infectious progeny virus. This suggests that nsp10 is involved in an as yet unidentified step of virion biogenesis.
Coronaviruses encode large replicase polyproteins which are proteolytically processed by viral proteases to generate mature nonstructural proteins (nsps) that form the viral replication complex. Mouse hepatitis virus (MHV) replicase products nsp3, nsp4, and nsp6 are predicted to act as membrane anchors during assembly of the viral replication complexes. We report the first antibody-mediated Western blot detection of nsp6 from MHV-infected cells. The nsp6-specific peptide antiserum detected the replicase intermediate p150 (nsp4 to nsp11) and two nsp6 products of approximately 23 and 25 kDa. Analysis of nsp6 transmembrane topology revealed six membrane-spanning segments and a conserved hydrophobic domain in the C-terminal cytosolic tail.
Many groups of plus-stranded RNA viruses produce additional, subgenomic mRNAs to regulate the expression of part of their genome. Arteriviruses and coronaviruses (order Nidovirales) are unique among plus-stranded RNA viruses for using a mechanism of discontinuous RNA synthesis to produce a nested set of 5′- and 3′-coterminal subgenomic mRNAs, which serve to express the viral structural protein genes. The discontinuous step presumably occurs during minus-strand synthesis and joins noncontiguous sequences copied from the 3′- and 5′-proximal domains of the genomic template. Nidovirus genome amplification (“replication”) and subgenomic mRNA synthesis (“transcription”) are driven by 13 to 16 nonstructural proteins (nsp's), generated by autocatalytic processing of two large “replicase” polyproteins. Previously, using a replicon system, the N-terminal nsp1 replicase subunit of the arterivirus equine arteritis virus (EAV) was found to be dispensable for replication but crucial for transcription. Using reverse genetics, we have now addressed the role of nsp1 against the background of the complete EAV life cycle. Mutagenesis revealed that nsp1 is in fact a multifunctional regulatory protein. Its papain-like autoprotease domain releases nsp1 from the replicase polyproteins, a cleavage essential for viral RNA synthesis. Several mutations in the putative N-terminal zinc finger domain of nsp1 selectively abolished transcription, while replication was either not affected or even increased. Other nsp1 mutations did not significantly affect either replication or transcription but still dramatically reduced the production of infectious progeny. Thus, nsp1 is involved in at least three consecutive key processes in the EAV life cycle: replicase polyprotein processing, transcription, and virion biogenesis.
The nsp2 replicase protein of porcine reproductive and respiratory syndrome virus (PRRSV) was recently demonstrated to be processed from its precursor by the PL2 protease at or near the G1196|G1197 dipeptide in transfected CHO cells. Here the proteolytic cleavage of PRRSV nsp2 was further investigated in virally infected MARC-145 cells by using two recombinant PRRSVs expressing epitope-tagged nsp2. The data revealed that PRRSV nsp2 exists as different isoforms, termed nsp2a, nsp2b, nsp2c, nsp2d, nsp2e, and nsp2f, during PRRSV infection. Moreover, on the basis of deletion mutagenesis and antibody probing, these nsp2 species appeared to share the same N terminus but to differ in their C termini. The largest protein, nsp2a, corresponded to the nsp2 product identified in transfected CHO cells. nsp2b and nsp2c were processed within or near the transmembrane (TM) region, presumably at or near the conserved sites G981|G982 and G828|G829|G830, respectively. The C termini for nsp2d, -e, and -f were mapped within the nsp2 middle hypervariable region, but no conserved cleavage sites could be definitively predicted. The larger nsp2 species emerged almost simultaneously in the early stage of PRRSV infection. Pulse-chase analysis revealed that all six nsp2 species were relatively stable and had low turnover rates. Deletion mutagenesis revealed that the smaller nsp2 species (e.g., nsp2d, nsp2e, and nsp2f) were not essential for viral replication in cell culture. Lastly, we identified a cellular chaperone, named heat shock 70-kDa protein 5 (HSPA5), that was strongly associated with nsp2, which may have important implications for PRRSV replication. Overall, these findings indicate that PRRSV nsp2 is increasingly emerging as a multifunctional protein and may have a profound impact on PRRSV replication and viral pathogenesis.
Coronaviruses express two very large replicase polyproteins, the 16 autoproteolytic cleavage products of which collectively form the membrane-anchored replication complexes. How these structures are assembled is still largely unknown, but it is likely that the membrane-spanning members of these nonstructural proteins (nsps) are responsible for the induction of the double-membrane vesicles and for anchoring the replication complexes to these membranes. For 3 of the 16 coronavirus nsps—nsp3, nsp4, and nsp6—multiple transmembrane domains are predicted. Previously we showed that, consistent with predictions, nsp4 occurs in membranes with both of its termini exposed in the cytoplasm (M. Oostra et al., J. Virol. 81:12323-12336, 2007). Strikingly, however, for both nsp3 and nsp6, predictions based on a multiple alignment of 27 coronavirus genome sequences indicate an uneven number of transmembrane domains. As a consequence, the proteinase domains present in nsp3 and nsp5 would be separated from their target sequences by the lipid bilayer. To look into this incongruity, we studied the membrane disposition of nsp3 and nsp6 of the severe acute respiratory syndrome coronavirus and murine hepatitis virus by analyzing tagged forms of the proteins expressed in cultured cells. Contrary to the predictions, in both viruses, both proteins had their amino terminus, as well as their carboxy terminus, exposed in the cytoplasm. We established that two of the three hydrophobic domains in nsp3 and six of the seven in nsp6 are membrane spanning. Subsequently, we verified that in nsp4, all four hydrophobic domains span the lipid bilayer. The occurrence of conserved non-membrane-spanning hydrophobic domains in nsp3 and nsp6 suggests an important function for these domains in coronavirus replication.
Autophagy, a catabolic pathway that delivers cellular components to lysosomes for degradation, can be activated by stressful conditions such as nutrient starvation and endoplasmic reticulum (ER) stress. We report that thapsigargin, an ER stressor widely used to induce autophagy, in fact blocks autophagy. Thapsigargin does not affect autophagosome formation but leads to accumulation of mature autophagosomes by blocking autophagosome fusion with the endocytic system. Strikingly, thapsigargin has no effect on endocytosis-mediated degradation of epidermal growth factor receptor. Molecularly, while both Rab7 and Vps16 are essential regulatory components for endocytic fusion with lysosomes, we found that Rab7 but not Vps16 is required for complete autophagy flux, and that thapsigargin blocks recruitment of Rab7 to autophagosomes. Therefore, autophagosomal-lysosomal fusion must be governed by a distinct molecular mechanism compared to general endocytic fusion.